A large set of genome-wide markers and a high-throughput genotyping platform can facilitate the genetic dissection of complex traits and accelerate molecular breeding applications. Previously, we identified about 0.9 million SNP markers by sequencing transcriptomes of 27 diverse alfalfa genotypes. From this SNP set, we developed an Illumina Infinium array containing 9,277 SNPs. Using this array, we genotyped 280 diverse alfalfa genotypes and several genotypes from related species. About 81% (7,476) of the SNPs met the criteria for quality control and showed polymorphisms. The alfalfa SNP array also showed a high level of transferability for several closely related Medicago species. Principal component analysis and model-based clustering showed clear population structure corresponding to subspecies and ploidy levels. Within cultivated tetraploid alfalfa, genotypes from dormant and nondormant cultivars were largely assigned to different clusters; genotypes from semidormant cultivars were split between the groups. The extent of linkage disequilibrium (LD) across all genotypes rapidly decayed to 26 Kbp at r2 = 0.2, but the rate varied across ploidy levels and subspecies. A high level of consistency in LD was found between and within the two subpopulations of cultivated dormant and nondormant alfalfa suggesting that genome-wide association studies (GWAS) and genomic selection (GS) could be conducted using alfalfa genotypes from throughout the fall dormancy spectrum. However, the relatively low LD levels would require a large number of markers to fully saturate the genome.